Method for producing addressed ligands matrixes on a support

ABSTRACT

A method for fabricating matrices of addressed ligands on a carrier. In the method, an element is used such as a reservoir filled with ligand and containing an electrode to deposit and electrochemically fix the ligand to the conductive carrier. The ligand may be an oligonucleotide or a peptide, and fixing may be obtained by electrocopolymerisation of this oligonucleotide or peptide carrying a pyrrole group at 5′ with pyrrole.

This is a 371 of PCT/FR00/00289, filed Feb. 8, 2000.

TECHNICAL FIELD

The subject of the present invention is a method for producing matricesof addressed ligands on a carrier.

The ligands may be natural or synthetic products having biologicalactivity or an affinity for biological or other molecules, for examplepeptides, oligonucleotides, receptors or other molecules of biologicalinterest. Matrices of this type may find numerous applications, inparticular for the detection and identification of constituents inbiological samples and for screening molecule libraries. Such matricesmay in particular be matrices of oligonucleotide probes.

PRIOR ART

In the past few years several methods have been developed for producingmatrices of this type. Three methodologies are known in which addressingis made either by photochemical route, or by mechanical route, or byelectrochemical route.

In the document by Fodor S. et al, Science, 1991, 251, pages 767–773 [1]a method is described for making a matrix of oligonucleotides byphotochemical addressing. According to this method, a carrier is usedfunctionalised by functional groups protected by photolabile protectorgroups; these protector groups are then removed by radiation through amask on the sites which are to be coupled to the molecules of biologicalinterest, then these molecules are coupled to the de-protectedfunctional groups.

This mode of photochemical addressing has the disadvantage of requiringa large number of different masks to carry out all the couplingoperations.

The documents: Khrapko K. R. et al, DNA Sequence—I.DNA Sequencing andMapping, 1991, volume 1, pages 375 to 388 [2] and GB-A-2 319 838 [3]describe a method for producing matrices by mechanical addressing. Indocument [2] a carrier is used which is coated with a polyacrylamide gelthat is activated by substituting certain amide groups by hydrazidegroups. The oligonucleotides activated in aldehyde form are then fixedto the hydrazide groups by micropipetting the oligonucleotide solutionsonto the sites to which they are to be coupled.

In document [3] a carrier is used which is functionalised by reagentgroups and coupled to identical biological molecules. The carrier isthen cut into individual plaques each one corresponding to the couplingof a molecule and then several plaques carrying different molecules atdesired sites are subsequently assembled on a plate.

The use of these mechanical addressing techniques has the disadvantageof having to bring the molecule to be fixed directly to the site to beaddressed. Therefore the size of the site cannot be smaller than thesize of the drop of dispensed reagent. Also, the process requires twophases which are respectively a dispensing phase and then a covalentattachment phase. Also the carrier has to be modified such that acovalent bond may be formed between the carrier and the molecule to befixed.

The documents: Livache T. et al, Nucleic Acids Res., 1994, 22, 15, pages2915–2921 [4] and WO-A-94/22889 [5] describe electrochemical addressingtechniques to produce matrices of biological products.

In this case a carrier is used which comprises several electrodes andthese electrodes are used to fix the biological molecules byelectrochemical route. For this purpose, the carrier fitted with itselectrodes is immersed in a solution containing the molecule to befixed, and by activation of the desired electrodes they are coated withthe molecule by electrochemical route. On this account, the deposits ofmolecules can only be made in successive manner. Moreover, it isnecessary to use a carrier carrying electrodes that can be individuallyaddressed, therefore complex systems that are possibly multiplexed.

The subject of the present invention is precisely a method for producingmatrices of biological products on a carrier, which remedies thedisadvantages of the above-mentioned methods and with which it ispossible in addition to conduct the addressing and fixing of thebiological molecule in a single step, without requiring priorfunctionalisation of the carrier.

DESCRIPTION OF THE INVENTION

For this purpose, the invention puts forward a method for producing amatrix containing at least one ligand fixed by electrochemical route toa conductive carrier or to conductive zones of a carrier, in which atleast one element is used able to dispense the ligand or ligands coupledto an electropolymerisable monomer serving as electrode to achieveelectrically assisted synthesis of a polymer carrying the ligand orligands on the conductive carrier or on the conductive zones of thecarrier.

According to the invention, an element is therefore used as electrodewhich is able to dispense the ligand or ligands. This element may bemade up of a reservoir containing the ligand coupled to theelectropolymerisable monomer and comprising a conductive part, or it maysimply be formed of an electrode in the form of a wire or needle which,after immersion in a container containing the ligand to be fixed coupledto the electropolymerisable monomer, is charged with this ligand bycapillarity.

By using an electrode formed of said element according to the invention,it is possible to place the ligand in contact with the conductivecarrier or the conductive zones of the carrier, then to fix it directlyto the conductive carrier (or the conductive zone) by electrochemicalactivation, for example by setting up a potential difference or bygenerating a current between the conductive carrier (or the conductivezone) and the element acting as electrode.

Therefore the dispensing and fixing of the ligand to the carrier isconducted in a single step.

According to a first embodiment of the invention, said element comprisesa reservoir filled with the ligand and comprising an insulatingdispenser nozzle and at least one electrode arranged in said reservoir,said nozzle being in direct contact with the conductive carrier or atleast one conductive zone of the carrier, during the fixing operation.

The nozzle may in particular be a capillary tube which is directlyplaced on the conductive surface.

According to a second embodiment of the invention, said elementcomprises a reservoir filled with ligand, and comprising a conductivedispenser nozzle, the contact between the conductive nozzle and theconductive carrier or at least one conductive zone of the carrier beingassured via a drop of ligand leaving the nozzle during the fixingoperation.

In this case, the conductive nozzle is not in contact with theconductive carrier or the conductive zone. As previously, the conductivenozzle may be formed of a capillary tube.

According to a third embodiment of the invention, said element is formedof an electrode in wire or needle form, charged externally with ligandcoupled to the electropolymerisable monomer, the contact between theelectrode and the conductive carrier or a conductive zone of the carrierbeing assured during the fixing operation by a drop of ligand withheldby the electrode.

In the different embodiments described above, the reservoir generallycontains a solution of ligand to be fixed and reagent(s) that mayoptionally be needed to ensure fixing of the ligand by electrochemicalroute.

According to the invention, the electrochemical fixing of the ligand ismade in particular by coupling it to an electropolymerisable monomer. Inthis case, the solution may contain the ligand coupled to theelectropolymerisable monomer, the electropolymerisable monomer andoptionally a doping agent.

The elctropolymerisable monomer may in particular be one of thosedescribed by Emr S. and Yacynych A., Electroanalysis, 1995, 7, pp.913–923 [7]. They may belong to two categories, those leading toconductive polymers such as pyrrole, aniline, thiophene. and theirderivatives, and those leading to insulating polymers such asderivatives of phenyl or benzene.

In this case, fixing of the ligand is achieved byelectrocopolymerisation of the monomer and of the ligand coupled to themonomer.

The ligand may for example be an oligonucleotide, a nucleotide, an aminoacid or a peptide.

Said method of electrochemical fixing is described in document [5] forligands which are an oligonucleotide or a nucleotide.

In this latter case, after conducting fixation, the chain of the fixedoligonucleotide or nucleotide can be lengthened through application ofconventional synthesis methods for oligonucleotides by successivecoupling of the desired nucleotides, but by conducting electrochemicalde-protection of the last nucleotide fixed.

In respect of peptides, it is possible to use the same technique tolengthen the chain of the peptide by coupling the desired amino acids.

The use of the electrodes described above to achieve the depositing andfixing of a ligand by electrochemical route has the followingadvantages:

-   -   The depositing and fixing procedure is carried out in a single        step and it is very rapid.    -   This technique is easy to implement since it simply uses a        mechanical depositing technique, for example transfer using a        micropipette, but it is coupled to the space resolution        possibilities of electrochemistry.    -   With this technique it is possible to carry out several deposits        in parallel mode.    -   Also, this method does not require the use of modified carriers        or which carry individually addressable electrodes.

For carriers in conductive material, these may be made entirely in anelectrically conductive material or they may be made of an insulatingmaterial coated with a layer of conductive material.

The conductive materials which can be used may be of different types,they may for example be metals such as gold, silver and platinum, orconductive oxides such as indium and tin oxide (ITO), carbon orconductive organic polymers.

If the carrier comprises conductive zones, these may be made in theconductive materials cited above and arranged on an insulating carrier.

The insulating carrier may for example be in glass, silicon or plasticmaterial. It is also possible to use a carrier in a conductive materialwhose conductive zones are delimited by depositing an insulatingmaterial on the surface of the conductive material.

According to the invention, the conductive zones may be electricallyinterconnected or electrically addressable either individually or ingroups so that they can be activated separately.

The method of the invention may be implemented such as to fix identicalor different ligands on different conductive sites of the carrier.

In this case, simultaneous or successive fixing of identical ordifferent ligands may be made using several elements respectivelydispensing identical or different ligands. In this case, at least two ofthe elements may be grouped together to form a print head.

According to one variant of the invention, successive fixing is made ofat least two different ligands to different sites of the carrier using asingle element but by changing the ligand dispensed by this element atleast once.

In all the embodiments described above, the main advantage lies in theligand dispensing-coupling process which enables the production ofcarriers carrying addressed molecules in extremely fast manner.

A further subject of the invention is a device for producing a matrix ofligands on a conductive carrier or on conductive zones of a carrier,comprising:

-   -   at least one ligand dispensing means provided with a conductive        part,    -   means for connecting firstly the conductive carrier or the        conductive zones of the carrier, and secondly the conductive        part of the dispensing means to an electric generator, and    -   means for positioning and/or moving the carrier and/or the        dispensing means, relative to one another and to place them in        contact such as to make several deposits of ligands on the        carrier at different sites.

According to the invention, the dispensing means may comprise areservoir containing the ligand and at least one electrode arranged insaid reservoir and forming the conductive part of said means.

According to one particular arrangement, the device comprises severalligand dispensing means assembled in the form of a print head.

According to one variant of embodiment, the device for producing amatrix of ligands on a conductive carrier or conductive zones of acarrier, comprises:

-   -   an electrode in the form of a wire or needle able to be charged        externally with said ligand,    -   means for connecting firstly the conductive carrier or the        conductive zones of a carrier, and secondly the electrode to an        electric generator, and    -   means for positioning and/or moving the carrier and/or the        electrode relative to one another such as to make several        deposits of ligands on the carrier at different sites.

Other characteristics and advantages of the invention will becomeclearer on reading the following description which is evidently givenfor illustrative purposes and is non-restrictive, with reference to theappended drawings.

SHORT DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of an element comprising a ligand dispensingreservoir and at least one electrode to fix the ligand to a conductivecarrier.

FIG. 2 shows an element similar to the one in FIG. 1 to achieve fixingof a ligand to a conductive carrier provided with conductive zones thatare electrically interconnected.

FIG. 3, on an enlarged scale, shows the nozzle of the dispensingreservoir in FIG. 1, to carry out fixing of the ligand on a carriercomprising multiplexed conductive zones.

FIGS. 4A and 4B illustrate the necessary steps to achieve fixing of aligand to a conductive carrier using an electrode in wire form.

FIG. 5 shows a dispensing element, fitted with a fluid inlet and outletto ensure its filling and draining, between two different ligand fixingoperations.

FIG. 6 is a diagram of a print head comprising several reservoirs fordispensing identical or different ligands.

DETAILED DISCLOSURE OF THE EMBODIMENTS

FIG. 1 shows the first embodiment of the invention in which as electrodean element is used comprising a reservoir 1 filled with the ligand to befixed and comprising a dispensing nozzle 1 a. Inside reservoir 1 arearranged a counter-electrode 3 made in platinum or gold for example, anda control electrode 5.

The reservoir may contain a sufficient volume of reagent to carry out acertain number of deposits, which may for example reach one thousand.

In this first embodiment shown in FIG. 1, a conductive carrier 7 is usedwhich may comprise a glass substrate coated with a gold layer.

This figure shows the deposits 9 made with said reservoir by moving thecarrier along directions x and y for example between two deposits. Ifthe nozzle 1 a of the reservoir, in the form of a capillary tube forexample, is made in an insulating material it can be placed on theconductive carrier 7 and, by setting up a difference in potential orcurrent between conductive surface 7 and the counter-electrode 3, it ispossible to obtain deposits 9 which are fixed to the conductive surface7 by electric impulse. In this case, the size of the deposits 9 isdetermined by the size of the reservoir/carrier interface located in thelines of the electric field between the electrode and the conductivesurface. This interface must be as small as possible to reduce the sizeof the deposit obtained.

The reservoir in FIG. 1 may also comprise a nozzle 1 a in conductivematerial. In this case, fixing of the ligand present in the reservoir ismade by contacting the conductive surface 7 with the electrode formed bynozzle 1 a by means of a drop leaving nozzle 1 a. In this case the sizeof the deposits is also adjusted by the interface between the liquid andthe conductive surface located in the lines of the electrical field.

The resolution of the deposits 9 may be improved by using a carrier asshown in FIG. 2 formed of interconnected conductive zones. In FIG. 2 thesame references have been used as in FIG. 1 to designate the reservoir 1fitted with its nozzle 1A, a control electrode 5 and a counter-electrode3. In this case the carrier is formed of an insulating carrier providedwith conductive zones 13 insulated from each other but electricallyinterconnected. These conductive zones may be made in gold on a glass orsilicon substrate for example. In this case, deposits 9 are obtained bydispensing the ligand above the conductive zones, but only theconductive zones in contact with the ligand can be coated with thelatter. Therefore the size of the deposits is adjusted by the size ofthe conductive zones 13.

In this case, the conductive carrier used in fact only comprises asingle electrode; this immensely simplifies its production and the costsinvolved may be very low since simple sheets of plastic material coatedwith conductive material may be used.

The use of a network of conductive zones makes it possible to reduce thesize of the deposits 9, but not to increase the density of the matrix.This density is directly dependent upon the size of the interfacebetween the capillary nozzle 1 a and the carrier and it is limited bythe size of the nozzle.

It is nonetheless possible to increase the density of the matrix byusing a carrier comprising conductive zones forming multiplexedelectrodes, as shown in FIG. 3.

FIG. 3 illustrates the nozzle 1 a of the reservoir 1 in FIGS. 1 and 2 onan enlarged scale and part of an insulating conductive carrier 11provided with conductive zones 13 which are separately connected tomeans for applying a potential or current so that they can be activatedseparately. In this case, the size of the deposits is determined by thesize of the activated conductive zones 12 as shown in the case in FIG.3. The other conductive zones which are in contact with the ligandcannot lead to fixing of the ligand since they are not electroactivated.In this manner, it possible to simultaneously achieve high spaceresolution and strong matrix density.

In FIGS. 4A and 4B another embodiment of the invention is shown in whichthe element able to dispense the ligand is formed by an electrode 15 inwire form.

In this case, a conductive carrier 7 can be used as shown in FIG. 4A. Tomake a ligand deposit, the electrode 15 is firstly immersed in acontainer 17 containing the ligand to be fixed and the electrodewithholds a drop 19 of this ligand. The electrode containing the drop 19of ligand is then brought above conductive carrier 7 as shown in FIG. 4Bmaking electric contact by means of drop 19. By applying an electricimpulse between electrode 15 and the conductive carrier 7 the formationof deposits of ligand is obtained.

After this operation, the electrode 15 is rinsed in a rinsing tank 21 sothat it can be used again to make another deposit 9 either with the sameligand or with another ligand.

When this type of electrode is used, the resolution of the deposits maybe lower but in this case the possible rinsing of electrode 15 is adeterminant advantage.

The method of the invention is of great interest since it provides thepossibility firstly of using a very small volume of reaction medium andtherefore of economising the molecules of biological interest to becoupled. Also, the size of the deposits made on the carrier may beadjusted whereas in mechanical addressing methods involving conventionalchemical activation methods the size of the deposits could not be lessthan 50, even 100 μm.

According to the invention, the size of the deposits can be very easilyreduced not by reducing the size of the drop which is difficult inpractice, but by reducing the surface of the zone that can beelectroactivated. The resolution of the deposits is optimised throughthe fact that only the electrode/carrier interface located in the linesof the electric field can be activated; that is to say that if a dropspills outside this zone, its content will not be fixed to theconductive surface.

Therefore if the diameter of the interface between the nozzle 1 a andthe conductive carrier is 200 μm, and if a conductive zone is used whoseside measurement is only 10 μm, only this conductive zone may be coatedwith the molecules of biological interest.

According to the invention, it is possible to make deposits 9 ofdifferent ligands on a carrier. This may be achieved by successivelyfixing at least two different ligands to different sites of the carrierusing a single element and by changing the ligand dispensed by saidelement. In this case, the deposits may be made successively, either bychanging the content of reservoir 1 of the elements shown in FIGS. 1 and2, or by using the electrode in FIG. 4 which is immersed in differentreagents. It is also possible to use a fixed reservoir provided withligand adding and evacuation means, that is to say comprising a fluidinlet and outlet system for the ligand so as to change the content ofthe reservoir without having to move it.

FIG. 5 illustrates said embodiment of reservoir 1 provided with a fluidinlet 1 b and an outlet 1 c.

Evidently, it is also possible in order to make deposits 9 of identicalor different ligands, to use several elements such as those shown inFIGS. 1 and 4. These elements may optionally be assembled to form aprint head as shown in FIG. 6.

In FIG. 6 it can be seen that the print head contains a first reservoirR1 filled with a ligand P1, a second reservoir R2 filled with a ligandP2 and a third reservoir R3 filled with a ligand P3. With a multiplehead of this type it is possible to make three simultaneous deposits 9of ligands P1, P2 and P3 respectively on the conductive surface 7.

It is specified that the deposits may be made in an inert atmosphere orin an electrochemically neutral liquid medium which, if possible, isnon-miscible with the reaction medium contained in the reservoir.

After the depositing phase, the carrier may be rinsed and used inconventional manner.

The following examples illustrate the production of matrices ofoligonucleotides or peptides using oligonucleotides or peptides carryinga pyrrole group which are fixed to a conductive carrier bycopolymerising them with pyrrole by electrochemical route using themethod described in document [5]: WO-A-94/22889.

EXAMPLE 1

1-Production of Carriers Carrying Oligonucleotides

The conductive carriers used are glass plates coated with a layer ofchromium (for adherence) and a continuous layer of gold of 0.5 μm. Thislayer is connected to the “working electrode” outlet of an EGG 283potentiostat.

Two different oligonucleotides carrying a pyrrole group at 5′ arecopolymerised on these carriers.

They were synthesized using the method described by Livache et al in[5].

To fix these oligonucleotides to the carrier, a reaction medium is usedcontaining 0.1 M LiClo4, 20 mM pyrrole and 1 μM oligonucleotide carryinga pyrrole group at 5′.

This solution is added to a reservoir in polypropylene of cone shapewhich contains a platinum counter-electrode (CE) connected to thepotentiostat. This reservoir is easily filled using a micropipette whosevolume may vary from 50 to 1000 μl reaction medium. The tip of this conehas a diameter of approximately 0.8 mm. Finer or larger cones can beused for other volumes of reagent.

The tip of the cone is placed in contact with the conductive surface andthe copolymer is made by cyclic voltametry (from −0.35 to +0.85V/CE atthe rate of 100 mV/s). The charge recorded is used to determine thethickness of the polymer formed. After the formation of this firstdeposit, the cone is emptied, rinsed then filled with a new reactionmedium containing another oligonucleotide. The conductive plate is moved(table x/y/z) and the same copolymerisation operation is conducted onanother area of the conductive surface enabling the production of adeposit carrying another oligonucleotide sequence.

In this manner two matrices are prepared solely comprising pyrM5oligonucleotides and two matrices solely comprising pyrCPoligonucleotides.

It is checked that the matrices of oligonucleotides so obtained have thedesired properties for detecting complementary oligonucleotides byhybridisation.

2-Hybridisation of Oligonucleotides and Detection.

The complementary oligonucleotides tested are the following:

-   -   biotinylated complementary M5: bio_(comp)M5;    -   biotinylated complementary CP: bio_(comp)CP.

The hybridisation of the complementary oligonucleotides is conducted inPBS buffer (Sigma containing 0.5 M NaCl, 100 μg/ml salmon sperm DNA(Sigma), 10 mM EDTA and 10 nM of complementary biotinylatedoligonucleotide. Hybridisation is conducted at 45° C. in a volume of 20mm for 15 min. Quick rinsing in PBS/NaCl is made. Detection of thehybrids is then carried out after incubation in PBS/NaCl solutioncontaining 0.1 mg/ml R phycoerythrine (Molecular Probe). Fluorescence isdetected using a cold camera (Hamamatsu) mounted on an epifluoresencemicroscope. The results are expressed as shades of grey.

A spot of polypyrrole approximately 0.8 mm in diameter is observed whosefluorescent intensity is reported below:

-   -   oligonucleotide on pyrM5 carrier hybridised with bio_(comp)M5:        110    -   oligonucleotide on pyrM5 carrier hybridised with bio_(comp)CP: 5    -   oligonucloetide on pyrCP carrier hybridised with bio_(comp)M5: 7    -   oligonucloetide on pyrCP carrier hybridised with bio_(comp)CP:        84

Good hybridisation specificity is observed with a high signal/noiseratio.

EXAMPLE 2

The same operating method is followed as in example 1 to preparematrices of pyrM5 and pyrCP oligonucleotides but using as conductivecarrier a carrier in plastic material coated with indium and tin oxide(ITO).

The results obtained with these matrices for the detection ofbiotinylated complementary oligonucleotides are the following:

-   -   oligonucleotide on pyrM5 carrier hybridised with bio_(comp)M5:        95    -   oligonucleotide on pyrM5 carrier hybridised with bio_(comp)CP: 5    -   oligonucleotide on pyrCP carrier hybridised with bio_(comp)M5: 7    -   oligonucleotide on pyrCP carrier hybridised with bio_(comp)CP:        105

EXAMPLE 3

In this example, the same operating method as in example 1 is followedto prepare a matrix of pyrM5 oligonucleotides on a carrier in goldsupported by glass but as counter-electrode a platinum wire is usedcharged with reaction medium instead of the reservoir fitted on theinside with a platinum electrode.

As shown in FIG. 4A, the platinum wire 15 is charged with reactionmedium by immersion in a reservoir 17 containing this medium. The wirecarrying the drop 19 is then brought to the carrier until contact ismade with the drop. The electrochemical impulse is then made. The wireis lifted away and rinsed in water. Other deposits are made in the samemanner. In this way deposits of approximately 1 mm in diameter areobtained and intense fluorescence is visible when the matrix is used toconduct hybridisation of the complementary oligonucleotide. The resultsobtained are the following:

-   -   oligonucleotide on pyrM5 carrier hybridised with bio_(comp)M5:        400    -   oligonucleotide on pyrCP carrier hybridised with bio_(comp)CP:        10

EXAMPLE 4

In the same manner, peptides may be deposited. Pyrrole-peptides aresynthesised using the procedure described by T. Livache et al, Biosensorand Bioelectronics 13, (1998) 629–634 [6]. They are deposited followingthe usual procedure ( ). The two peptides ACTH (18-39) and ACTH (11-24)are then detected by the biotinylated antibodies Mab (34-39) and Mab(18–24) respectively.

Fluorescence results after incubation with streptavidin phycoerythrineare the following:

Peptide ACTH 18–39 with Mab 34–39 640 Peptide ACTH 18–39 with Mab 18–24510 Peptide ACTH 11–24 with Mab 34–39 10 Peptide ACTH 11–24 with Mab18–24 470

CITED REFERENCES

-   [1] Fodor S. et al, Science, 1991, 251, pp. 767–773.-   [2] Khrapko K. R. et al, DNA Sequence—I.DNA Sequencing and Mapping,    1991, vol. 1, pp. 375–388.-   [3] GB-A-2 319 838.-   [4] Livache T. et al, Nucleic Acids Res., 1994, 22, 15, pages    2915–2921-   [5] WO-A-94/22889.-   [6] T. Livache et al, Biosensors and Bioelectronics 13, (1998),    pages 629–634.-   [7] Emr S. and Yacynych A, Electroanalysis, 1995, 7, pp. 913–323.

1. A method for electrochemically fixing a matrix of deposits of aligand on a plurality of sites of a conductive carrier or of conductivezones of a carrier, said method including use of an electrode and acarrier laterally movable relative to one another, said electrode beingconfigured to distribute a discrete volume of a solution containing saidligand coupled to an electropolymerisable monomer, said methodcomprising the steps of: a) positioning the electrode above one of saidplurality of sites, b) distributing with the electrode on said one ofsaid plurality of sites said discrete volume of a solution andsimultaneously circulating an electric current from said electrode tosaid site to polymerize said electropolymerisable monomer so as toelectrochemically fix the ligand on said one of said plurality of sites,d) repositioning the electrode over another of said plurality of sitesvia lateral movement relative to the carrier, and e) repeating saidsteps of distributing and simultaneously circulating an electricalcurrent so as to form said matrix.
 2. The method according to claim 1,wherein the electrode comprises: a reservoir containing the solution,and a conductive part.
 3. The method according to claim 2, wherein thereservoir comprises: means for filling said reservoir with said solutionand evacuating said solution from said reservoir.
 4. The methodaccording to claim 1, wherein the electrode comprises one of a wireelectrode and a needle electrode configured to be charged externallywith said solution of ligand coupled to the electropolymerisablemonomer, and said step of distributing comprises establishing a contactbetween the one of a wire electrode and a needle electrode and the oneof said plurality of sites by a drop of said solution withheld on theone of a wire electrode and a needle electrode.
 5. The method accordingto claim 1, wherein the conductive zones of a carrier are formed ofzones of conductive material arranged on an insulating carrier.
 6. Themethod according to claim 5, wherein the zones of conductive materialare electrically interconnected.
 7. The method according to claim 5,wherein the plurality of conductive zones comprises: a conductivematerial chosen from the group consisting of gold, silver, platinum,indium and tin oxide (ITO), carbon, and conductive organic polymers. 8.The method according to claim 1, wherein the solution comprises: adoping agent.
 9. The method according to claim 1, wherein theelectropolymerisable monomer is pyrrole.
 10. The method according toclaim 1, wherein the step of electrochemically fixing the ligandcomprises: fixing the ligand by electro-copolymerisation of both theelectropolymerisable monomer and the ligand coupled to theelectropolymerisable monomer.
 11. The method according to claim 1,wherein the ligand comprises: one of a nucleotide, an oligonucleotide,an amino acid, and a peptide.
 12. A method for producing a matrix ofdeposits of different ligands electrochemically fixed on a plurality ofsites of a conductive carrier or of conductive zones of a carrier, eachsite configured to receive a predetermined one of a plurality ofdifferent ligands, said method including the use of several electrodesand a carrier movable relative to one another, said electrodesconfigured to distribute a discrete volume of one of a plurality ofsolutions, each of said plurality of solutions containing one of aplurality of ligands coupled to an electropolymerisable monomer, saidmethod comprising the steps of: a) simultaneously or successivelypositioning two or more of said several electrodes above a correspondingtwo or more of said plurality of sites, b) distributing on each of saidtwo or more of a plurality of sites said discrete volume of one of aplurality of solutions, and simultaneously circulating an electriccurrent to said one of a plurality of sites to polymerize theelectropolymerisable monomer within said one of a plurality of solutionsso as to electrochemically fix the ligand within said one of a pluralityof solutions on said two or more of said plurality of sites, d)repositioning said two or more of said several electrodes above a secondcorresponding two or more of said plurality of sites over another ofsaid plurality of sites via lateral movement relative to the carrier,and e) repeating said steps of distributing and simultaneouslycirculating and electric current so as to form said matrix.
 13. Themethod according to claim 12, wherein said two or more of said severalelectrodes comprises: at least two electrodes arranged together in aprint head.
 14. The method according to claim 12, wherein each of saidseveral electrodes comprises: a reservoir configured to contain saiddiscrete volume, and a conductive part.
 15. The method according toclaim 14, wherein the reservoir comprises: means for filling saidreservoir with said solution and evacuating said solution from saidreservoir.
 16. The method according to claim 12, wherein each of saidplurality of several electrodes comprises one of a wire electrode and aneedle electrode configured to be charged externally with said solutionof ligand coupled to the electropolymerisable monomer, and said step ofdistributing comprises establishing a contact between the one of a wireelectrode and a needle electrode and a corresponding site by a drop ofsaid solution withheld on the one of a wire electrode and a needleelectrode.
 17. The method according to claim 12, wherein the conductivezones of the carrier are formed of zones of a conductive materialarranged on an insulating carrier.
 18. The method according to claim 17,wherein the zones of the conductive material are electricallyinterconnected.
 19. The method according to claim 17, wherein theconductive material is chosen from the group consisting of gold, silver,platinum, indium and tin oxide (ITO), carbon, and conductive organicpolymers.
 20. The method according to claim 12, wherein each of saidplurality of solutions comprise: a doping agent.
 21. The methodaccording to claim 12, wherein the electropolymerisable monomer ispyrrole.
 22. The method according to claim 12, wherein the step ofelectrochemically fixing the ligand comprises: fixing the ligand byelectro-copolymerization of both the electropolymerisable monomer andthe ligand coupled to the electropolymerisable monomer.
 23. The methodaccording to claim 12, wherein the ligands are chosen from the groupconsisting of nucleotides, oligonucleotides, amino acids and peptides.